Neuropeptide Y (NPY) is a 36 amino acid alpha-amidated peptide, widely found throughout the nervous system. Co-existence of neuroactive peptides and classical transmitters in neurons has been frequently observed. The proposed studies examine the mechanisms of regulation of synthesis, secretion and storage of multiple co-localized neuroactive substances. The model system chosen for the studies is the rat superior cervical ganglion (SCG) since SCG neurons can be maintained in tissue culture and produce norepinephrine (NE), acetylcholine (ACh) and NPY. The initial aim of this proposal is to determine the biosynthetic pathway of NPY. The identification of the cDNA sequence of the human NPY precursor has provided the means to probe NPY biosynthesis. Studies will determine the synthetic pathway from pro-NPY to NPY and other product peptides in rat SCG neurons. The specific proteolytic cleavages of pro-NPY and any intermediate biosynthetic steps will be detailed. Studies will examine likely co- and post-translational modifications of pro-NPY including amidation and phosphorylation. The developmental profile for NPY in rat SCG will be determined and compared to that of NH and ACh. The second major goal is to examine neuronal control of co-existing neuroactive substances in SCG. Investigations will explore the effect of known regulatory factors on the content and synthetic rate of NPY, NE, and ACh measured simultaneously. In particular, studies will evaluate changes i neuronal activity and in the culture medium on transmitter and peptide levels and synthetic rates and on NPY mRNA. The SCG model system also enables study of the activity of two related biosynthetic enzymes, peptidyl-glycine-Alpha-amidating monooxygenase (PAM) and dopamine-Beta-hydroxylase (DBH) and enables comparison of PAM activity with amidated NPY product under various conditions. NPY and NE will also be localized immunohistochemically and the extent of co-localization under various culture conditions will be determined. NPY is a potent constrictor of vascular smooth muscle. In addition the localization of NPY is sympathetic ganglion and othr grainstem sites suggest a role in neural control of blood pressure and other autonomic function. It is expected that these studies will provide further insight into the regulation of NPY in neurons and to the special requirements of neurons with co-existing neuroactive substances.